Abstract

Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.

Elicitation of heat shock proteins (HSPs) by pustulan is due to β-1,6-glucan

Neutrophils were cultured with opsonized beads for 2 hours at 37 °C in A and F. (A) Endo-β-1,6-glucanase reduces the induction of HSPs by pustulan. Beads were coated with an equivalent amount of pustulan (pus) or endo-β-1,6-glucanase digested pustulan. Induction of HSPs with enzyme treated pustulan is relative to that with untreated. The data represent the average of three experiments with standard deviation. (B) Pustulan chromatographed on Biogel P6 column. (C) Pustulan digested first with endo-β-1,6-glucanase and run on a P6 column generated a large and a small peak. The small peak represents a tiny fraction of the original pustulan that was resistant to enzymatic digestion (Vo). (D) The large peak in C was shown by thin layer chromatography to be the expected degradation products, gentiobiose and gentiotriose. Lane 1 contains standard oligosaccharides (G to G5) as controls. Lane 2 is pustulan spiked with glucose (G). Lane 3 is endo-β-1,6-glucanase digested pus. Ori = origin. (E) Chromatography of deacetylated pustulan. The insert is an overlay of the Vo from C and E. (F) Deacetylation of pustulan followed by digestion with endo-β-1,6-glucanase eliminates induction of HSPs. Induction of HSPs in deacetylated pustulan or deacetylated pustulan digested with endo-β-1,6-glucanase is relative to that with untreated pustulan.

β-1,6-glucan stimulates phagocytosis and production of reactive oxygen species (ROS) in neutrophils

Polybead polystyrene 6 μm microspheres (beads) were coated with an equivalent amount of the indicated β-glucans and then opsonized. (A) β-1,6-glucan stimulates phagocytosis. Phagocytosis was assessed by time-lapse microscopy for beads that were coated with laminarin (a, and b), or pustulan (c and d). The images at a and c were taken at time 0. Images b and d were taken after culturing with neutrophils for 40 minutes. (B) β-1,6-glucan stimulates phagocytosis. Phagocytosis was assessed by Fluorescence Activated Cell Sorting (FACS) by the change in side scatter for neutrophils with (a) untreated beads, (b) beads coated with β-1,3-glucan from Candida (c) beads coated with laminarin (lam, β-1,3-glucan), (d) beads coated with glucan from barley (bar), (e) beads coated with pustulan (pus, β-1,6-glucan) (f) soluble pustulan. (C) β-1,6-glucan stimulates ROS production. ROS production was assayed by FACS using DHR123. β-1,3-glucan shows only a modest stimulation.

Beads were untreated (Beads), or coated with equivalent amount of laminarin (lam, β-1,3-glucan), or pustulan (pus, β-1,6-glucan). Following opsonization, the beads were suspended in 2% SDS 1M ammonium hydroxide buffer and incubated at 37 °C for 1 hour. The supernatant solution was loaded on 4–20% acrylamide SDS gel. The migration of the molecular weight protein standards is indicated. (A) The gel was incubated with silver stain, and the bands were extracted for analysis by mass spectrometry. (B) Western analysis was performed using monoclonal antibodies directed against (a) the alpha or (b) the beta chains of C3.

(A) Phagocytosis of pustulan- coated beads was assessed by FACS, by the change in side scatter. Serum was untreated (a), or incubated for 5 minutes at 37 °C with 1 mg (quantified by phenol- sulfuric acid method) of soluble laminarin (lam) (b) or pustulan (pus) (c). (B) Reactive oxygen species production in response to pustulan- coated beads was assayed by FACS using DHR123. Serum was untreated (red), incubated with soluble laminarin (green), or with pustulan (blue). (C) C3 deposition on pustulan- coated beads was eliminated by preincubation of the serum by soluble pustulan but not laminarin. C3 deposition was assayed by Western analysis using monoclonal antibodies directed against the (a) alpha or (b) the beta chains of C3. Serum was preincubated with soluble pustulan (1), laminarin (2), or was untreated (3). The molecular weight protein standard is indicated. (D) Preincubation of serum with soluble pustulan reduces Candida killing. Serum was untreated, or preincubated with soluble pus or lam prior to Opsonization of Candida. Candida viability was assayed using XTT following incubation of 30 minutes with neutrophils.

β-1,6-glucan is required for efficient phagocytosis of Candida albicans, production of ROS, and expression of HSPs

Candida albicans cells were heat killed, digested with an endo-β-1,6-glucanase, and opsonized. (A) β-1,6-glucan is required for efficient phagocytosis. Phagocytosis was assessed by Fluorescence Activated Cell Sorting (FACS) by the change in side scatter. (B) β-1,6-glucan is required for efficient ROS production. ROS production was assayed by FACS using DHR123. (C) β-1,6-glucan is required for induction of HSPs. HSPs induction was determined by quantitative real-time PCR. Results for β-1,6-glucanase digested Candida were normalized to undigested Candida. The data represent the average of two experiments with standard deviation.